Many common birth defects result from defective morphogenesis. Our primary working hypothesis is that morphogenesis depends on cell polarity, which is established and maintained by cell surface-cytoskeletal interactions that are sensitive to asymmetric cell-cell contacts. Our objective is to investigate this hypothesis using mouse blastocyst formation as an experimental model of mammalian morphogenesis. Blastocyst formation entails two interrelated events, 1) cavitation and 2) differentiation of trophectoderm and inner cell mass (ICM). This project focuses on the morphogenetic details of cavitation, which is critical to trophectoderm/ICM differentiation. I have proposed the Metabolic Cavitation Model to explain cavitation and production of nascent blastocoele fluid. The unique features of this model are: 1) Production of nascent blastocoele fluid depends on cytoplasmic polarity consisting of the asymmetric distribution of organelles (mitochondria and lipid droplets) that accumulate along apposed cell surfaces. 2) Maintenance of cytoplasmic polarity depends on: a. properties of the plasma membrane that distinguish free- from apposed cell surfaces; b. cell surface-cytoskeletal interactions that are sensitive to asymmetric cell-cell contacts; c. transcellular ion currents that enter free (apical) surfaces and leave apposed (basolateral) surfaces of blastomeres having asymmetric cell-cell contacts (the outer blastomeres of the morula). 3) Water in nascent blastocoele fluid results from beta-oxidation of lipid droplets. The Specific Aims will test these features of the Metabolic Cavitation Model. Plant lectins will be used (immunofluorescence/immunoperoxidase techniques) to selectively label specific plasma membrane domains together with treatments to disrupt cytoskeletal elements and cell surface-cytoskeletal associations. Effects on organelle distribution will be quantitated by morphometric analyses at the electron microscopic (EM) level. Isolated blastomeres will be subjected to electric fields to obtain information on how molecules are segregated to specific plasma membrane domains. Lipid catabolism during cavitation will be quantitated by morphometric analyses at the EM level.